1. Field of the Invention
The present invention relates to the field of separating a peptide from a resin to which the peptide is bonded, following solid phase peptide synthesis.
2. Description of the Background Art
Solid phase peptide synthesis involves assembling amino acids into a peptide of any desired sequence while one end of the chain is anchored to an insoluble support. The insoluble support is a synthetic polymer which bears reactive groups. The amino acid which forms the C-terminal reside of the peptide to be synthesized is converted to a derivative in which its amino group is protected by a labile protecting group. The derivative of the C-terminal amino acid is coupled to the reactive polymer. A reagent is applied to the protected aminoacyl polymer to remove the labile blocking group from the amino acid residue. The reagent must not harm the link of the C-terminal residue to the polymer in any way. Moreover, if the amino acid attached to the polymer contains a side-chain reactive functional group, that functional group must be blocked by a stable blocking group which will remain completely intact throughout the synthesis, but which can be removed finally to yield the free peptide. Following removal of the labile protecting group, the next amino acid is coupled to the aminoacyl polymer by use of a suitable coupling reaction. Again, the .alpha.-amino group must be protected with a labile protecting group. This cycle of deprotection and coupling is then repeated with each amino acid which is to be incorporated into the peptide chain. Finally, after the entire blocked peptide has been assembled on the polymer support, a different type of reagent is applied to cleave the peptide from the polymer and allow it to be dissolved. The blocking groups which have protected side-chain functional groups must also be removed, and usually are chosen so that they can be removed simultaneously with cleavage of the peptide from the resin.
One reagent for cleavage of peptide from the resin at the end of the synthesis is anhydrous liquid hydrogen fluoride (HF). HF cleavage is generally done at 0.degree. C. for 30 minutes. Such conditions will generally cleave the peptide effectively from the resin and remove all side-chain blocking groups. The HF then is removed, e.g., under vacuum, and the cleaved peptide then is separated from the resin.
A common problem associated with HF cleavage of resin-peptide following solid phase peptide synthesis is side reactions caused by prolonged contact of the peptide with HF. In order to avoid "bumping", or a sudden surge of HF/resin slurry, the process is carried out very slowly, thereby prolonging the exposure of peptide to HF and causing the above-noted side reactions. The problem is even more pronounced in a large scale cleavage, e.g., greater than one liter, when a large quantity of HF cannot rapidly be removed after a proper reaction time has elapsed. Furthermore, constant monitoring and adjustment of vacuum level are required to control the process.
After removal of HF, the peptide is extracted from the resin with appropriate solvent(s). However, the low solubility of some peptides makes it difficult to completely recover the peptide from the resin, resulting in a lower yield.
There thus remains a need in the art for improved methods of separating peptides from resins so as to reduce or eliminate side reactions.